FUNCTIONAL GENE DISCOVERY USING RNA INTERFERENCE-BASED GENE SILENCING

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 一般来说，目前的抗生素靶向病原体而不是宿主特异性生化途径，以确保患者的毒性较低，药物不良反应较少。这种策略的缺点是它经常导致多药耐药（MDR）细菌群体的发展。 大型制药公司已经开始将重点从开发新类别的抗生素转移到其他更有利可图的药物靶点，例如治疗慢性疾病的药物。 为了防止潜在的公共卫生危机，在打击传染病，我们认为，鉴定的重要宿主蛋白质的病原体在感染过程中的目标，可以提供可行的候选新药开发，以抵消发病机制。 为了鉴定这些候选宿主蛋白，我们正在使用RNA干扰（RNAi）进行全基因组功能丧失高通量筛选（HTS）。RNAi筛选已应用于多项研究，寻求新的治疗靶点，以对抗癌症和慢性（HIV）或重复性（流感）病毒感染。 在最近的一项RNAi研究中，发现激酶PBK/AKT 1可调节沙门氏菌和结核分枝杆菌在人类细胞中的细胞内生长，这导致开发出具有抗生素特性的AKT激酶抑制剂。这项原理证明研究表明，基于RNAi的功能丧失测定不仅可以发现物种特异性抗菌治疗靶标，还可以发现具有广谱抗菌活性潜力的候选物。 我们的目标是确定宿主蛋白的两种不同的病原体，耶尔森氏菌属和伯克霍尔德氏菌属，这有一个共同的致病机制，抑制宿主细胞信号级联阻断细胞对感染的反应，III型分泌系统（TTSS）。 TTSS包括细菌效应蛋白和将其注射到宿主细胞中所必需的蛋白。我们重点关注鼠疫耶尔森氏菌（鼠疫和肺鼠疫的病原体）和类鼻疽伯克霍尔德氏菌（传染病类鼻疽的病原体），因为它们的高毒力和潜在的威胁，如果故意释放武器化的MDR菌株的社会破坏。致病性耶尔森氏菌通过抑制由GTP结合的Rho家族蛋白（RhoA，Rac-1，Cdc 42）的受体触发激活引发的肌动蛋白聚合来抵抗宿主巨噬细胞和PMN白细胞的吞噬作用。这些宿主蛋白通过Yop效应蛋白的同步作用而失活：（1）YopT蛋白酶，（2）YopO/YpkA激酶，和（3）YopH磷酸酪氨酸磷酸酶（Rosqvist et al 1990，Andersson et al 1995，Bliska et al 1995，Fallman et al 1995，Grosdent et al 2002）。 对于伯克霍尔德氏菌，已经发现来自TTSS-3的基因编码与来自沙门氏菌属的TTSS结构“针”蛋白和分泌效应物高度同源的蛋白，表明这些同源Bsa蛋白也可能在调节伯克霍尔德氏菌发病机制中起作用。 耶尔森氏菌和伯克霍尔德氏菌效应蛋白被认为与多个宿主蛋白靶相互作用，以使病原体能够存活并定殖宿主。 不幸的是，这些病原体特异性靶向的宿主蛋白质在很大程度上仍然没有特征。我们希望我们的方法，使用高通量的siRNA为基础的敲低策略将开始，以确定宿主蛋白质，特别是针对耶尔森氏菌和伯克霍尔德氏菌属，提供有价值的分子见解TTSS介导的毒力在宿主中的机制，并作为基础的设计新的抑制剂治疗，阻断感染。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. In general, current antibiotics target the pathogen rather than host-specific biochemical pathways to ensure lower toxicity and less adverse drug effects in the patient. A drawback of this strategy is that it often leads to development of multi-drug resistant (MDR) bacterial populations. Major pharmaceutical companies have begun to shift focus from development of new classes of antibiotics to other more profitable drug targets, such as those that treat chronic conditions. To forestall a potential public health crisis in combating infectious disease, we posit that identification of essential host proteins that are targeted by pathogens during infection can provide viable candidates for novel drug development to counteract pathogenesis. To identify these candidate host proteins, we are performing genome-wide loss-of-function high-throughput screens (HTS) using RNA interference (RNAi). RNAi screens have been applied in multiple studies seeking new therapeutic targets to counteract cancer and chronic (HIV) or repetitive (influenza) viral infections. In a recent RNAi study, the kinase PBK/AKT1 was found to regulate intracellular growth of Salmonella and Mycobacterium tuberculosis in human cells, which led to development of AKT kinase inhibitors that exhibit antibiotic properties. This proof of principle study demonstrates that RNAi-based loss of function assays can uncover not only species-specific anti-bacterial therapeutic targets but also candidates with potential for broad spectrum antimicrobial activity. We aim to identify host proteins that are targeted by two different pathogens, Yersinia spp and Burkholderia spp, which share a common pathogenic mechanism for inhibition of host cell signaling cascades to block cellular response to infection, the type III secretion system (TTSS). TTSS includes both the bacterial effector proteins and the proteins necessary for their injection into the host cells. We focused on Yersinia pestis (the etiological agent of bubonic and pneumonic plague) and Burkholderia pseudomallei (the causative agent of the infectious disease melioidosis) because of their high virulence and potential threat for social devastation in case of intentional release of weaponized MDR strains. Pathogenic Yersinia spp resist phagocytosis by host macrophages and PMN leukocytes through inhibition of actin polymerization initiated by receptor-triggered activation of GTP-bound Rho family proteins (RhoA, Rac-1, Cdc42). These host proteins become inactivated by the synchronous action of the Yop effector proteins: (1) the YopT protease, (2) the YopO/YpkA kinase, and (3) the YopH phosphotyrosine phosphatase (Rosqvist et al1990, Andersson et al 1995, Bliska et al 1995, Fallman et al 1995, Grosdent et al 2002). For Burkholderia, genes from TTSS-3 have been found to encode for proteins that are highly homologous to both TTSS structural "needle" proteins and secreted effectors from Salmonella spp., indicating that these homologous Bsa proteins may also play a role in regulating Burkholderia pathogenesis. The Yersinia and Burkholderia effector proteins are thought to interact with multiple host protein targets to enable pathogen survival and colonization of the host. Unfortunately, the host proteins specifically targeted by these pathogens remain largely uncharacterized. We expect that our approach to use a high-throughput siRNA-based knock-down strategy will begin to identify host proteins that are specifically targeted by Yersinia and Burkholderia spp, provide valuable molecular insights into the mechanisms of TTSS-mediated virulence in the host, and serve as the basis for design of novel inhibitor therapeutics that block infection.